In this project we propose producing, and testing in vivo, a recombinant protein that should sustainably reduce plasma low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein secreted in the liver, plays a key role in maintaining cholesterol homeostasis by regulating cell-surface levels of the low-density lipoprotein receptor (LDLR), a plasma membrane glycoprotein that removes LDL-C particles from the plasma. Levels of circulating PCSK9 in human populations vary over a >100- fold range, and there is a positive correlation between PCSK9 levels and levels of LDL-C (and between PCSK9 and coronary artery disease events). Statins are widely prescribed as therapy for hypercholesterolemia (high cholesterol). A therapy that would down-regulate PCSK9 should act as an adjunct to statins in maintaining healthy levels of LDL-C, especially in individuals with familial hypercholesterolemia. A protein fragment containing the epidermal growth factor-like repeat AB (EGF-AB) domains of the LDLR binds to PCSK9 and blocks the ability of PCSK9 to deplete LDLR on hepatocytes. The EGF-AB fragment, however, is unstable in vivo, and thus impractical as a therapeutic. In this project we propose to produce, and test in vivo, a stable decoy molecule incorporating EGF-AB, which should deplete plasma PCSK9 and sustainably reduce plasma LDL-C. Using a plant expression system we will produce a series of recombinant proteins comprised of EGF- AB variants fused to the Fc of human IgG1. One variant will incorporate the wild-type EGF-AB sequence, but the others will contain single amino acid mutations found in the LDLR of individuals with hypercholesterolemia (and some non-natural mutations). It is expected that some of these variants will bind more tightly to PCSK9 and thus better block its ability to deplete LDLR. The EGF-AB variants will first be assayed for their ability to interfere with the binding of labeled LDLR extracellular domain to PCSK9. The variants with the greatest activity will then be tested in a cellular LDL uptake assay to determine how well they inhibit the LDLR-lowering activity of exogenously added PCSK9. The two best EGF-AB-Fc variants will be tested for their ability to lower LDL-C in transgenic mice expressing elevated levels of human PCSK9. PUBLIC HEALTH RELEVANCE: Coronary artery disease is the leading cause of death worldwide. The primary causal factor in the pathogenesis of coronary artery disease is an elevated plasma level of low-density lipoprotein cholesterol (LDL-C). A receptor decoy based on a protein fragment of the low-density lipoprotein receptor fused to IgG Fc is expected to dramatically lower LDL-C and reduce the risk of coronary artery disease.